This document contains proprietary information that Profectus BioSciences requests not be released to persons outside the Government, except for purposes of review and evaluation. Abstract The enhanced safety, stability, and accelerated product development generally provided by DNA vaccination make it an appealing approach to develop prophylactic and therapeutic HIV vaccines. Unfortunately, successful DNA vaccination of primates requires multiple inoculations with undesirably large doses of plasmid. Genetically encoded adjuvants could provide the immune stimulating and dose-sparing effects necessary to realize a practical DNA-based vaccine for HIV. Our adjuvant approach exploits the RIG-1 signaling cascades that recognize infection with viral pathogens and trigger innate immune responses. We have constructed dominant-positive versions of RIG-1-like helicases and their adaptor protein (IPS-1) that mimic viral infection when transfected into cells as plasmid DNA. Our objective is to determine whether these plasmid expressed RIG-1 based constructs will adjuvant an HIV DNA vaccine in mice to a greater extent than the "bench mark" adjuvant IL-12. We will pursue our objective through the following specific aims: (1) Identify an adjuvant or adjuvant combination that induces maximal B and T cell mediated immune responses against HIV GP160;(2) Demonstrate that the lead adjuvant provides enhanced protection in a Vaccinia-HIV challenge model. The co-formulation that is immunogenic and protective in mice will be evaluated in additional mice and primate studies as components of an advanced HIV DNA vaccine in follow-on Phase II SBIR applications. 2 PUBLIC HEALTH RELEVANCE: The objective of this project is to develop novel adjuvants for an HIV DNA vaccine. Such a vaccine/adjuvant combination is needed to combat the HIV epidemic.